Dr Rosie Staniforth

Research Precis

Until very recently, our understanding of the causes behind neurodegenerative diseases has been very limited. The outbreak of BSE (commonly referred to as mad cow disease) and the rise of Alzheimer’s has meant that there is increasing pressure to obtain a cure. These two diseases are characterised by the accumulation of fibrous material called "amyloid" in the brain. This substance has been shown to consist of naturally occurring proteins assembled into this unnatural mass. Whether or not this protein-based material is the direct cause for deterioration of brain tissue in patients suffering from these diseases is a matter of some controversy. However, it is clear to all that these proteinaceous fibres are closely associated with the pathology: mutations in genes coding for these proteins cause the early onset of the disease and premature death. Another cause for hope is that this pattern of events may be common to almost all neurodegenerative diseases, including Parkinson’s and Huntington’s, and a large number of other diseases including certain forms of cancer, diabetes and stroke. It is exciting to think that understanding the mechanism of amyloid formation may be a step towards a universal cure since, so far, therapies have only been targeted at alleviating symptoms but cannot prevent or halt the onset of these devastating diseases.

As a biochemist, my interest is in studying the assembly of "normal" proteins into amyloid fibres at a molecular level (fig.1). A first consideration when tackling such a problem is that, unlike other polymers, protein molecules exist as unique three-dimensional structures in vivo (referred to as their folded state). In amyloidogenesis, proteins with totally different folds form fibres with very similar properties suggesting there may be common, structurally analogous intermediates on the assembly route. This also means that considerable conformational changes (unfolding) of the protein molecules are likely to occur either before or after association.

A well-characterised protein with a number of advantageous properties is cystatin C, a protease inhibitor which is associated with a disease causing recurrent stroke (Human Cystatin C Amyloid Angiopathy). I have identified a number of different conformers of this protein which could all be candidate precursors for the assembly process. These include partially folded states of the monomeric protein and a "domain-swapped" dimer. The latter species (fig. 2) is the result of two molecules of cystatin coming together and exchanging part of their chains by unfolding then refolding into a conformation made up of two cystatin-folds, where each protein chain contributes half its constituent amino acids to one fold and half to the other. This is the first time this kind of intertwining of protein chains is observed for an amyloidotic protein. Such a process of assembly results in the formation of extremely stable oligomers and has often been speculated as a convenient mechanism for amyloid fibre formation. Structural characterisation of the different forms of cystatin from monomer to dimer to fibre stretches the boundaries of current technology. We are using a combination of techniques including dynamic NMR (nuclear magnetic resonance) spectroscopy and high-resolution electron microscopy.

Identifying different states of the protein is only the first step in characterising the mechanism of amyloid formation: each species must be put in context within a sequence of molecular events, i.e. a pathway needs to be defined. To do this, we have set up a screen to identify optimum conditions for observing fibre formation in vitro. Time courses will be recorded when the reaction is initiated from a number of different states of the protein including the dimeric form. This will not only establish a mechanism for the first time but it will also provide an experimental system which can be used to screen for potential therapeutic drugs.